Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Fig. S3 ATP hydrolysis activity of Xcc NAMPT.ATP hydrolysis activities of Xcc NAMPT and its H229A mutant were evaluated by the NADH-coupled detection system.Control groups consisting of LYK5 (lysin motif receptor kinase 5, which lacks the ability to hydrolyze ATP alone 2 ) and buffer were utilized in the study.The experiments were performed three times independently.Fig. S8 Active site comparison of NAMPT in its unbound (apo) state with its bound state in complex with either NAM or NMN.The apo structure is shown in green, the NAM-NAMPT complex in magenta, and the NMN-NAMPT complex in cyan.To distinguish between Tyr 14 and Phe 177, which are located in different protomers of a dimer, a single comma was used as a label for Tyr 14.    S5.S4.The primers (pLAFR-F/R) for the green box were used to detect the presence of the pLAFR3 vector.The primers (XC0719-F/R) for orange box were used to detect the presence of Nampt.The primers (XC1067-F/R) for blue box were used to detect the presence of Nads.

Fig. S4
Fig. S4 Crystal packing may affect substrate binding.a NAMPT monomer with an empty active site.The α11-13 helices are labeled within a square area and the missing helices are labeled in gray.The apo structure displayed here is derived from the diamond-shaped crystal used for soaking substrates, and this apo structure has only one monomer in each asymmetric unit.b NAMPT monomer in complex with NMN or NAM.The carbon atoms of NMN are shown in cyan, and the two NAM molecules in green and yellow, respectively.The α11-13 helices are labeled within a similar square area.c Packing scheme of NAMPT dimers in the crystal."Empty" and "Occupied" indicate the empty site and substrate-bound site, respectively.The packing details are shown in the box.The dashed area indicates the motifs affected by packing.

Fig. S5
Fig. S5 Dimerization is essential for enzyme activity of Xcc NAMPT.a Gel filtration and SDS-PAGE analysis of purified 6× His-tag Xcc NAMPT.The elution volumes of the molecular weight standards are marked on top of the gel filtration curve, and the molecular weight of a NAMPT dimer is also indicated.The upper right panel shows the SDS-PAGE analysis of the peak fraction obtained from the gel filtration.b-c Complementation of inactive mutant pairs.The pairs of mutants were mixed and incubated for 24 hours at 4°C (b) or left untreated (c) prior to activity assessment.For the reaction, NAMPT variants at a concentration of 25 nM were incubated with 10 μM NAM, 50 μM PRPP, and 2.5 mM ATP.The reactions were carried out for 15 minutes at 37℃, and the quantification of NMN was achieved using the fluorescence assay.

Fig. S7
Fig. S7 Stereo diagram shows the structure of Xcc NAMPT in a complex with NAM.The catalytic site NAM is shown in green and the tunnel NAM in yellow.The NAM molecules are shown as sticks.

Fig. S9
Fig. S9 Substrate-velocity curve for the R373A+R180A mutant pair of Xcc NAMPT with NAM.To determine the kinetic parameters of NAM, a concentration of 0.3 nM mutant pair was used.The NAM concentration was varied from 0.1 to 4 μM.The error bars depicted on the plot indicate the standard deviation of measurements taken from three replicates.

Fig. S12
Fig. S12 Analysis of histidine phosphorylation on Xcc NAMPT.a-b Xcc NAMPT was digested with trypsin and analyzed by LC-MS/MS.The spectrum obtained revealed the presence of pHis at the canonical His229 site.The matched b and y ions, as indicated in the spectrum and the accompanying sequence diagram, provide confirmation of the phosphorylation event.a Analysis of Xcc NAMPT.b Analysis of ATP-treated Xcc NAMPT that was purified through gel filtration with the removal of ATP.

Fig. S14
Fig. S14 Substrate binding sites of type II PRTs and monomer structures of NAPRTs.a Multiple sequence alignment for key substrate binding motifs of Xcc NAMPT with other NAPRTs and QAPRTs with solved protein structures.Residues that interact with NAM, NA, and QA are marked with orange, blue, and green squares, respectively.Conserved residues in the consensus sequence are colored.b Schematic representation of three substrates (NAM, NA, and QA) of phosphoribosyltransferases.c The monomer structures of Xcc NAMPT and NAPRT.The conserved domains A and B are depicted in pale green and gray, respectively.d Comparison of the key residues in active sites from two types of NAPRTs.The database and accession codes of sequences and structures used in this figure are indicated in TableS5.

Fig. S15
Fig. S15 Phylogeny and structural analysis of type II phosphoribosyltransferases.The phylogenetic tree presented in this figure corresponds to Fig.7a, but with complete species names, PDB ID numbers, and Uniprot ID numbers labelled for each of the protein sequences used.

Fig. S16 NAM
Fig. S16 NAM or NMN binding stabilizes the conformation of two protein helices, α11 and α12. a Structural comparison of the NAM/NMN binding site and the empty site.Our analysis revealed several steps that occur during NAM (left panel) or NMN (right panel) recognition.We assigned a sequence number to each major step of the binding process: (1) Substrate entry, (2) Forced movement of Tyr 14', (3) Stabilization of Glu 228 (originally part of a flexible loop that converts into α11 after NAM/NMN binding) by Tyr 14' and Lys 15', (4) Restriction of the flexible loop by Glu 228 to a relatively fixed site, and (5) Formation of helix 11. b The flexible loop in the empty site may affect the formation of helix 12.In the empty sites of the NAM and NMN complex structures, Ala 227 from the flexible loop could potentially clash with Met 247 from helix 12 if the helix were to form normally.Disruption of the formation of helix 12 may, in turn, impede the formation of helix 11 since these helices appear to stabilize each other.

Fig. S17
Fig. S17 Superimposition of NAM and NAT molecules bound within the tunnel.The catalytic site NAM and the tunnel NAM are shown in green and yellow, respectively, while NAT is in white.The PBD ID of the NAT-complex structure is 7ENQ.

Fig. S19
Fig. S19 Structure similarity between a Xcc MFS transporter and a Chlamydia trachomatis NAD + transporter.
1561-bp DNA fragment of the ORF XC_0719 of Xcc strain; Tet r This study pRSFDuet1 Duet vectors are T7 promoter expression vectors; Kan r EMD Biosciences (Novagen) pRSFDuet1-Xcc NAMPT pRSFDuet1 containing a 1407-bp fragment of the ORF XC_0719 of Xcc strain; Kan r This study pRSFDuet1human NAMPT pRSFDuet1 containing a 1476-bp DNA fragment of the ORF human NAMPT; Kan r This study Rif r , Kan r , and Tet r indicate resistance to rifampicin, kanamycin, and tetracycline, respectively.

Table S1 Bacterial strains and plasmids
lacZα, sacB, mob site.Allelic exchange vector (Suicidal vector carrying sacB gene for mutagenesis); Kan r